While hydrogels offer a facile method for in situ delivery of cells, they are not conducive to simultaneously withstanding the large forces found in joints (requiring high moduli) and promoting stem cell differentiation (requiring low moduli). Moreover, a mismatch in mechanical properties between scaffold and the adjacent tissue can lead to mechanical destabilization and eventually degeneration in the surrounding joint tissue. This points to the need for a mechanically robust scaffold that can withstand normal joint loads. In osteochondral tissues, cells reside in their own niche and are largely protected from large forces by the extracellular matrix. The proposed tissue engineering solution lies in mimicking nature's solution to this complex problem. Specifically, we will decouple the structural (i.e., load-bearing) component from the cellular niche within our hydrogel design. A stiff and functionally graded, load-bearing structural hydrogel component will withstand large forces and transfer appropriate strains (i.e., mechanical signals) to each cellular niche. Independently, three cellular niches will capture chemistries and degradation appropriate to hyaline cartilage, calcified cartilage and bone. When combined with dynamic loading that transfers mechanical cues from the structural component to each cellular niche, stem cell mediated OC tissue regeneration will be achieved. Our approach is possible by the enabling technologies of digital projection photolithography and highly tunable photoclickable hydrogels. Thus the overarching hypothesis for this research is: a structurally stiff and functionally graded material embedded within a soft material containing stem cells supports normal joint loads, minimizes damage to the surrounding tissue, and promotes OC tissue regeneration. To test this hypothesis, we have outlined three specific aims. In specific aim #1, we will design architecturally-controlled 3D OC mimetic hydrogel materials to support stresses similar to native OC tissue in vivo and transfer appropriate strains to each layer of the OC mimetic hydrogel. We will test the ability of an acellular and mechanically stable OC mimetic hydrogel to minimize damage to tissue surrounding an OC defect in swine knees. In specific aim #2, we will investigate MSC differentiation and OC tissue regeneration when MSCs are incorporated in the soft cellular component that is designed with biochemical and mechanical cues appropriate to each OC niche and cultured in custom bioreactors that mimic aspects of the in vivo loading environment. In specific aim #3, degradable and mechanically stiff OC mimetic hydrogels with autologous MSCs will be implanted in a swine OC knee defect for 12 weeks and evaluated for engineered OC tissue and damage to tissues surrounding the defect. Upon completion of this project, we expect to have demonstrated a mechanically stiff hydrogel with encapsulated MSCs is capable of (a) withstanding large forces, (b) promoting stem cell mediated OC tissue regeneration and (c) maintaining the health of the tissue surrounding the defect. Long-term, we are developing a miniaturized and portable printing technology that will be easily accessible to surgeons via an arthroscopic platform.